Laboratory of Human Toxicology and Pharmacology (LHTP) - The Laboratory of Human Toxicology and Pharmacology (LHTP) provides technical and operational support for DCTD programs designed to increase the pace and accuracy of drug development in oncology and facilitate the entry of new chemical entities (NCEs) for cancer treatment into Phase 0 and I clinical trials. LHTP is an integrated, multi-disciplinary laboratory program that builds on the foundation of pharmacodynamics (PD) and toxicodynamics (TD) to predict the desirable and undesirable human drug effects that will likely be encountered during the clinical evaluation of both traditional cytotoxic as well as newer molecular target based small molecules and engineered therapeutic viruses. LHTP expertise in predictive human toxicology using in vitro models, pharmacodynamic assay development and clinical implementation, and preclinical drug formulation combine to humanize treatment regimens, therapeutic assessment, and interpretation of non-clinical models of cancer. The laboratory integrates this novel expertise to humanize treatment and assessment of mouse models of human disease in order to validate pharmacodynamic assays for use as primary endpoints of Phase 0 trials, which are conducted under exploratory IND guidelines from the FDA to confirm that a NCE works in man as it has in the preclinical models. The integrated approach includes a route of administration, dosage form, dose level, and clinical sampling procedures (e.g., anesthesia, needle, or excisional biopsy) that are directly relevant to a Phase 0 clinical trial. The LHTP introduced the concept and practice of completely humanizing preclinical models of cancer therapy, which led to the use of clinical biopsy procedures in human xenograft and orthotopic tumor models to prove the clinical readiness of a PD assay. LHTP took the lead in reverse translational research, to learn the clinical procedures of tissue collection and then transfer them to DTP/BTB. Comparing humanized and traditional preclinical study designs in cancer models over several years will yield the results required to determine whether this approach improves the correlation between nonclinical models and the clinic. The LHTP is organized into four specialized, but interrelated, sections that align with the technical support needs of DCTD: Pharmacodynamic Assay Development and Implementation (PADIS), Predictive Toxicology (PTS), Viral Vector Toxicology (VVTS), and Formulation Development (FDS). The laboratory director manages the overall scientific program of the LHTP and its coordination with DCTD operations, including coordinated interactions and assay transfers from LHTP to the National Clinical Target Validation Laboratory (NCTVL) operating at the NIH campus. Pharmacodynamic Assay Development and Implementation Section (PADIS) - Modern developmental therapeutic efforts for cancer utilize an understanding of signal transduction or differentiation pathways to identify new drug candidates that exert defined actions on the functional status of these targets. Some of these defined molecular effects will have therapeutic potential, leading to apoptosis, differentiation, or other desirable changes in the function of cancer cells. By coordinated use of the preclinical efficacy models of the Biological Testing Branch of DTP, it is possible to define the relationship between the extent of drug effect in the tumor and the level of drug exposure associated with these effects. Clinical confirmation of these preclinical findings would provide strong rationale for a full clinical development program, and the FDA recently authorized a new type of early clinical trial to explore microdoses of investigational agents prior to full dose finding and safety trials. This exploratory IND mechanism (x-IND) is relevant for multiple therapeutic classes, and is being used by the DCTD to conduct such confirmatory studies of molecular drug action on tumor target in a small number of patients prior to full clinical development (termed Phase 0 trials at the NCI). Failure of a NCE to affect molecular target during the Phase 0 trial would provide a rationale to halt its development. The mission of PADIS is to provide expert technical support to this DCTD initiative by developing and validating laboratory assays that quantify a drug's effect on a molecular target in a tumor (and, if possible, in surrogate tissues as well), and can be readily implemented in the clinical setting. PADIS uses preclinical models to develop and validate pharmacodynamic (PD) assays and companion procedures for tissue acquisition that will be used in the clinical trial without change. A unique aspect of the PADIS approach is the constraints imposed on the development and validation of PD assays by the radiological, surgical, and anesthesia procedures and practices that will eventually be used clinically to obtain the tissue specimens for the PD assays. This is a technical area that has been underappreciated in the past, and PADIS works closely with BTB to integrate anticipated medical procedures into the preclinical models and develop clinically ready assays. When they can be established as accurate reporters of drug action in tumor, surrogate organ compartments (such as peripheral blood mononuclear cells or bone marrow or buccal keratinocytes) are useful for less invasive monitoring of drug effects than biopsies. As the final step in PD assay development and implementation, PADIS is responsible for cross-training the Bethesda-based NCTVL staff in the new assays and then coordinating with NCTVL to transfer the validated method to the clinical laboratory for deployment in the x-IND clinical trial. PADIS plays a key technical role in the developmental stages of clinical pharmacodynamic projects of the DCTD and its collaboration with the CCR in Phase 0 clinical trials. These stages can be summarized as: rapid development and validation of sensitive, SOP-driven methodologies to quantify the impact of drug treatment on molecular target(s) in tumors; identification of normal tissue that is a surrogate for tumor, if possible (PBMCs, skin, saliva, buccal mucosa, etc); development and validation of clinically-transferable, SOP-driven procedures for tissue collection and processing that will provide evaluable specimens in the clinical setting; and oversee the transfer of validated PD assays to the NCTVL for deployment in clinical trials, and if necessary, contribute to assays of clinical specimens. Predictive Toxicology Section (PTS) - The Predictive Toxicology Section (PTS) was established to facilitate the maturation and validation of assays that have been shown to predict human toxicity, and deploy them in concert with pharmacokinetic (PK)/ pharmacodynamic (PD) programs in the DTP to facilitate development of NCEs more efficiently and accurately for potential advancement to clinical trials. Preclinical toxicology studies on potential cancer chemotherapeutic agents are generally conducted in two species of animals with the goals of defining the maximum tolerated dose (MTD), dose-limiting toxicities (DLT), schedule dependency of toxicity, reversibility of adverse effects, and a safe clinical starting dose (SD). These animal studies do not always permit detailed evaluations of molecular toxicity, especially in relation to the expected mechanism in man and the role of off-target effects in mediating toxicity. Furthermore, the in vivo data sometimes show discrepancies between species that create uncertainty about human safety and result in low starting doses in early clinical trials. The PTS is tasked with the development of in vitro tissue, cellular, and molecular assays that use normal target tissues of drug toxicity and clinically relevant endpoints (e.g., in vitro transaminitis) to evaluate potential human safety, predict tolerated human doses, and allow direct comparison of human and animal drug tolerance prior to clinical trials. This information can be used to include predicted human tolerated dose levels into studies by PADIS to validate PD assays at predicted clinical dose levels, increasing confidence that a target response to the drug will be detected clinically. The recent validation of in vitro bone marrow assays for neutropenia using rodent, canine, and human CFU-GM myeloid stem cells demonstrated the value of in vitro assays for predicting in vivo toxicities in animals and man. The PTS seeks to extend the application of the validated testing principles to other dose-limiting organ system toxicities, including pulmonary, hepatic, and cardiac toxicities. In addition, PTS is tasked with assisting the Toxicology and Pharmacology Branch, DTP in its evaluation of newly developed in vitro toxicity tests under its grant program entitled Innovative Toxicology Models for Drug Evaluation and the importing of promising assays into PTS for validation. The in vitro models of normal tissue response to adverse drug effects are expected to provide a simpler system than the in vivo models used for the discovery of off-target drug effects that can be circumvented via analoging and toxicodynamic markers of organ toxicity, which become candidates for valid assay development by PADIS and used by medical laboratories to monitor for early stages of drug toxicity prior to the development of symptoms. Newly developed in vitro assays are validated and then transferred to the VVTS for specialized applications in the assessment of human safety of engineered viral therapeutics (see below). These novel safety assessment tools can be deployed in the pre-IND or IND-directed setting. Viral Vector Toxicology Section (VVTS) - The primary mission of the VVTS is to provide support for preclinical development of anticancer viral vectors. The research efforts are currently focused on the investigation of human toxicity potential of novel anticancer viral vectors by using humanized in vitro assay systems, and the evaluation of oncolytic activity and tumor selectivity of cancer-targeting viral vector agents. The VVTS explores the potential application of newly developed toxicity tests in PTS to the in vitro human safety assessment of engineered viral therapeutics. Additional areas of research include the assessment of the feasibility and efficacy of anticancer vector-combination therapy, consisting of viral vector agents and other cancer treatments of different modalities (e.g., chemotherapy or radiation); the elucidation of the molecular basis of tumor selection and oncolysis by candidate viral vectors; identification and establishment of tumor-specific biomarkers of viral agent-susceptible cancers, which can be adopted for future in vivo applications; and the development of optimal viral burden quantification assays to monitor the replication of viral vectors in targeted (tumor) and non-targeted (normal) tissue compartments in conjunction with in vivo safety and/or efficacy evaluations. During the reporting period, VVTS established a new in vitro assay system to evaluate the hepatotoxic potential of adenovirus-based anticancer vectors using primary human hepatocytes, characterized differential hepatotoxicity profiles of recombinant adenoviral vectors developed as therapeutic agents under the RAID program, and began collaboration with Biological Testing Branch (BTB), DTP, to evaluate the in vivo efficacy of oncolytic reovirus type 3 Dearing strain in a human melanoma xenograft model. Formulation Development Section Many NCEs that show promising anticancer characteristics exhibit very low aqueous solubility, which creates technical difficulties during in vitro and in vivo evaluations. The Formulation Development Section (FDS) is tasked with development of preclinical formulations of these NCEs suitable for intravenous injection and oral administration to mice and dogs and for testing in vitro in cell cultures. The FDS is developing a novel, moderate-throughput strategy comprised of an initial empirical screen, using 25 mg of each NCE, to test for solubility in a dozen proven vehicles representing diverse solubilizing mechanisms, which have been used clinically. Suitability of these candidate pre-formulations for injection is proven by continued solubility when challenged with physiological fluids. It is expected that 1020% of poorly soluble NCEs will not be solubilized by conventional vehicles, and so the next step in the strategy is formulation testing of NCEs in novel pharmaceutical technology platforms, such as nanodispersions and emulsions, targeted nano-particles, and targeted carrier proteins. Although not all NCEs will have been formulatable by the end of the second step, this strategy is expected to achieve solubility and consistency requirements for in vitro studies of efficacy in the DTP screen and of in vitro human toxicity by PTS with a large majority of NCEs in the DTP pipeline. A large number of these formulated NCEs will also be suitable for intravenous and oral administration in animal models: BTB efficacy studies, BTB and TPB studies of PK and PD including validation of molecular PD assays by PADIS, and PTS assessment of toxicity via biomarkers (PTS) or clinical assessment. These preclinical formulations are intended to facilitate preclinical evaluation of developmental compounds, and are not necessarily the same as the formulated clinical product that may advance into clinical trials.